One case of bloodstream infection caused by Ureaplasma urealyticum / 中国热带医学

@#Abstract: Objective To analyze a case of bloodstream infection caused by Ureaplasma urealyticum after abortion in Anxi County Hospital, so as to provide basis for the clinical diagnosis and treatment. Methods　The diagnosis of Ureaplasma urealyticum in this patient with bloodstream infection was retrospectively analyzed. The basic clinical data and laboratory diagnosis data were collected, including the characteristics of blood culture curve, Wright staining of culture medium, drug sensitivity of Mycoplasma liquid identification, colony characteristics of solid medium, and the conclusion of targeted DNA sequencing. Through the comprehensive analysis of the above data, the rapid diagnosis of this case can be realized by optimizing the detection and diagnosis process. Results　The clinical manifestations of this patient were fever of 38.5 ℃, CRP:14.85 mg/L, WBC:14.33×109/L, NET: 85.40%, PCT: 0.12 ng/mL, IL-6: 665.6 pg/mL, positive after 3 days of blood culture, no bacteria were found in Gram stain, and sand-like purple bacteria were observed after adding Wright's stain. After inoculation in blood agar, Mycoplasma solid and liquid medium, no colonies were grown in blood agar, after 48 h and 5 d. On Mycoplasma A7 agar, the edge of brown fried egg colony was striature, and it could be identified as Ureaplasma urealyticum with the Mycoplasma ID & AST panel, which was resistant to quinolones and spectinomycin, but sensitive to macrolides, tetracyclines and lincomycin. Subsequent targeted DNA sequencing results were also confirmed for Ureaplasma urealyticum. Before receiving the report, clinical experience treatment with ceftriaxone metronidazole was used to fight infection with negative bacilli and anaerobic bacteria. Mycoplasma was not treated with targeted treatment. After 3 days, the patient's body temperature returned to normal, inflammation index decreased, and the patient asked to be discharged. Conclusions　At present, there are few reports of bloodstream infection caused by Ureaplasma urealyticum, and the lack of clinical understanding can easily lead to misdiagnosis and missed diagnosis. In order to improve the detection rate of Mycoplasma in blood culture, it is necessary to optimize the detection procedure of blood culture and provide accurate diagnosis and treatment basis for clinical practice. However, it is clear from this case that Mycoplasma bloodstream infection cases are self-limited infection and can recover by themselves without targeted treatment in patients with normal immunity. Therefore, it is very important to protect the immunity of patients.

Effect of age on reversible lens opacity and Na⁺-K⁺-ATPase expression induced by chloral hydrate in mice / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of age on the expression of Na⁺-K⁺-ATPase and acute reversible lens opacification induced by chloral hydrate in mice. <p>METHODS: Acute reversible lens opacification was induced by intraperitoneal injection of 4% chloral hydrate(400mg/kg)in 3-month-old(young group)and 24-month-old(old group)C57BL/6 mice. The lens opacification was graded at 10, 20, 30, 45, 60, 90, 120 and 150min after chloral hydrate injection. The histopathological changes of lens were observed by hematoxylin eosin staining, and the expression of Na⁺-K⁺-ATPase in lens was detected by immunohistochemistry. <p>RESULTS: The development of lens opacity is similar in young and old mice after chloral hydrate injection. The lens opacification in the young group appeared earlier, thicker and lasted longer than the old group. HE staining showed that many vesicles appeared in the cortex below lens epithelial cells(LECs), and the structure of superficial lens fiber cells were disordered after chloral hydrate injection. Immunohistochemical staining showed that the expression of Na⁺-K⁺-ATPase was positive in LECs and fibers. The expression of Na⁺-K⁺-ATPase in LECs were weak before chloral hydrate injection and up-regulated 45min after chloral hydrate injection in young and old groups. The up-regulation of Na⁺-K⁺-ATPase was stronger in the old group than in the young group. <p>CONCLUSION: Age may play a role in the acute reversible lens opacification induced by chloral hydrate in mice. The expression of Na⁺-K⁺-ATPase is involved in lens opacity induced by chloral hydrate.

Ganoderic acid A protects lens epithelial cells from UVB irradiation and delays lens opacity / 中国天然药物

A contributory role of oxidative stress and protection by antioxidant nutrients have been suspected in cataract formation. Ganoderic acid A (GAA), an effective lanostane triterpene, is widely reported as an antioxidant. The aim of this study is to investigate the potential effects of GAA on cataract formation. After lens epithelial cells (LECs) were exposed to UVB radiation for different periods, cell viability, apoptosis-related protein levels, malondialdehyde (MDA) and superoxide dismutase (SOD) activities were monitored. We found that cell viability, the Bcl-2/Bax ratio and SOD activity were increased, while Cleaved caspase-3 levels and MDA activity were decreased compared with those in UVB-impaired LECs after GAA treated. Furthermore, GAA activated PI3K/AKT in UVB-impaired LECs and effectively delayed the occurrence of lens opacity in vitro. In conclusion, these findings demonstrated that GAA exhibited protective functions in SRA01/04 cells and rat lenses against UVB-evoked impairment through elevating cell viability and antioxidant activity, inhibiting cell apoptosis, activating the PI3K/AKT pathway and delaying lens opacity.

Prevalence and associated factors for pterygium in rural people aged 50 years and above in Funing County,Jiangsu Province / 中华实验眼科杂志

Objective To investigate the prevalence and associated risk factors for pterygium among people aged 50 years and above in Funing County,Jiangsu Province.Methods A cluster random sampling method was performed,the subjects aged 50 years or above were randomly selected from 30 survey sites in Funing County,Jiangsu Province.Questionnaires,visual acuity tests,the examinations of eye surface,anterior segment,fundus examinations were conducted.Pterygium was diagnosed and graded clinically by slit lamp examination.The risk factors were acquired from questionnaires and analyzed by the multivariate logistic regression analysis.This study protocol was approved by Ethic Committee of Affiliated Hospital of Nantong University (NO.2010-05).Written informed consent was obtained from each subject prior to entering study cohort.Results A total of 6 145 persons aged 50 years and above were enumerated,and actually 5 947 (96.8％) participants were examined.Among them,1 950 cases were diagnosed as pterygium in either eye and 1 228 cases were diagnosed as pterygium in binoculus,which was equivalent to the 32.79％ [95％ confidence interval(CI):31.60％-33.98％] of pterygium in either eye and 20.65％ (95％ CI:19.62％-21.68％) in bilateral pterygium.Among 2467 male subjects,838 were diagnosed as pterygium (33.97％,95％ CI:32.10％-35.84％).Among 3480 female subjects,1 112 were diagnosed as pterygium (31.95％,95％ CI:30.40％-33.51％).There was no significant difference in the prevalence of pterygium between genders (P =0.135).Multivariate Logistic analysis showed that,older age (50 ～ ＜60 years:odds ratio [OR] =1.00;60 ～ ＜70 years:OR=1.54,P＜0.001;70 ～ ＜80 years,OR=1.83,P＜0.001;≥80 years:OR=1.99,P＜0.001),low educational level (no education:OR =1.00;＜primary:OR =0.87,P =0.031;primary education:OR =0.72,P =0.002;≥ secondary education:OR =0.63,P =0.002),farmer occupations (OR =1.34,P =0.020),and long outdoor work time (OR =1.13,P =0.026) were independent risk factors for pterygium.Gender,marriage,income,hypertension,diabetes,smoking and alcohol use history were not associated with pterygium (all at P＞0.05).Conclusions The prevalence of pterygium in Funing County is 32.79％ in people aged 50 years and above.The high prevalence of pterygium may be associated with older age,low education level and long outdoor work time.

Meta-analysis of prevalence of pterygium among people aged over 40 in China / 中华实验眼科杂志

Objective To evaluated the prevalence of pterygium among people aged ≥40 in China by using Meta analysis.Methods A literature retrieval was performed in China biology medicine disc (CBMdisc),China national knowledge infrastructure (CNKI),VIP database,WanFang database,PubMed,excerpta medica database (EMBASE),Web of Science from 1990 to 2016 according to designed searching strategy.Tailored quality evaluation standard of epidemiological research was used to evaluate the study quality of each included study.Stata 13.0 software was used to estimate the prevalence of pterygium,subgroup analysis of age,sex and region was also carried out.Results Thirty-six studies were involved in this Meta analysis,including 8 English researches and 28 Chinese researches.The random effect model was used to merge 36 research data.The prevalence of pterygium among people aged over 40 in China was 13.4％ [95％ confidence interval (CI):10.6％-16.5％].Subgroup analysis results showed that the prevalence of pterygium increased with age.The prevalence of pterygium in rural region was 15.3％ (95％ CI:12.1％-18.8％),which was higher than 4.0％ (95％ CI:2.4％-6.1％) in urban region.The prevalence of pterygium among rural people aged ≥ 40 was 12.1％ (95％ CI:8.6％-16.2％),which was lower than 14.7％ (95％ CI:10.5％-19.5％) among rural people aged ≥50.The highest prevalence of pterygium in different age and sex subgroups was in the west region of China,followed by the east region of China and the lowest was in the central region of China.Conclusion The prevalence of pterygium among people aged ≥ 40 in China was high.Over the past decade,the prevalence of pterygium were different among different ages,and places of residence.

Expression and significance of monoacylglycerol Oacyltransferase 2 (MOGAT2) in pterygium / 眼科新进展

Objective To investigate the expression and significance of monoacylglycerol O-acyltransferase 2 (MOGAT2) in the tissues of pterygium.Methods Immunohistochemistry staining methods were adopted to detect the expression of MOGAT2 in 54 patients of pterygia (primary in 50 patients and recurrence in 4 patients)and 18 patients of normal conjunctival tissues.The semi-quantitative integration method was used to analyze the strength of immunohistochemical expression,and the results were compared and statistically analyzed,and the relationship between the expression of MOGAT2 in the pterygium group and the factors such as age,sex,and clinical pathological stage was analyzed.Results Immunohistochemical staining showed that,in 54 patients of pterygium,MOGAT2 was strongly expressed in 8 patients,positive expression in 29,weakly positive expression in 8 and negative expression in 9,while in 18 normal conjunctival tissues,strongly positive expression of MOGAT2 was found 1 patient,positive expression in 6,weakly positive expression in 3,negative expression in 8,respectively.Rank-sum test showed that the difference in MOGAT2 expression in the pterygium and normal conjunctiva tissues was statistically significant(Z =-2.403,P =0.016).There was no significant difference in the expression of MOGAT2 between the different age and the sex patients with pterygium (all P ＞ 0.05),and there was no significant correlation of MOGAT2 expression with education,occupation,income,protection in outdoor work,smoking,drinking,blood sugar,blood pressure,primary and recurrence pterygium,but significant correlation with the clinical stage of pterygium,that is,MOGAT2 expression in advanced pterygium was overexpressed when compared with tumor tissues in quiescent stage (P ＜ 0.05).Conclusion MOGAT2 is highly expressed in pterygium tissues,and the expression in advanced pterygium was significantly upregulated,of which is related to the formation and development of pterygium.

Prevalence of uncorrected refractive errors of rural population aged ≥ 50 years in Funing county, Jiangsu province / 眼科新进展

Objective To investigate the prevalence of uncorrected refractive errors among urban population aged 50 years and above in Ftming county,Jiangsu province.Methods Survey research was conducted and randomly cluster sampling was used to select individuals aged ≥50 years for visual acuity test and eye examination in Funing county,Jiangsu province.The criteria of uncorrected refractive errors in this study was defined as an improvement of at least 0.2 log MAR (equivalent to 2 lines) in the best corrected visual acuity with the base vision ＜ 0.5 log MAR in daily life.The quantitative data were expressed as mean ± standard deviation,and t-test was used for comparison between groups,and while the count data were expressed as rate or composition ratio,and the x2 test was adopted for comparison between the groups.Logistic regression was used to examine the effect of possible factors (i.e.age and gender) on the prevalence of uncorrected refractive errors.Results A total of 6145 persons aged 50 years and above were enumerated and 5947 (96.8％) participants were examined,of whom 2388 had uncorrected refractive errors,with the prevalence of 40.2％.The prevalence of uncorrected refractive errors for myopia only,hyperopia only,astigmatism,and for hyperopia and astigmatism were 84.4％,84.2％,64.1％ and 100％,respectively.Moreover,the higher prevalence of uncorrected refractive errors presented in elderly person (OR =1.07,P ＜ 0.00l) and female (OR =1.38,P ＜ 0.001),and education was a protective factor for junior high school (OR =0.74,P =0.003) and high school (OR =0.55,P ＜ 0.001).Conclusion Uncorrected refractive errors presented high prevalence in rural population aged 50 years and above in Funing county,Jiangsu province,which are the leading cause of visual impairment.

Distribution of lanosterol synthase and lanosterol in cornea, lens and retina tissue of rats / 中华实验眼科杂志

Background Researches showed that triterpenoids,with a similar structure to lanosterol,has therapeutical effect on many systemic diseases,and lanosterol was determined to have a therapeutical effect on cataract recently.However,how the lanosterol plays effects on other eye diseases is still unelucidated.Understanding the distribution of lanosterol in ocular tissue is helpful for us to elucidate the relationship of lanosterol with eye diseases.Objective This study attempted to investigate the distribution of lanosterol synthase (LSS) and lanosterol in cornea,lens and retina tissue of rats and offer a basis for the targeting treatment of eye diseases.Methods Fifteen SPF male SD rats were sacrificed by excessive anesthesia to obtain the eyeballs.The relative expressions of LSS protein and gene in the cornea,lens and retina tissue of the rats were detected by Western blot and reverse transcription (RT)-PCR,respectively.Immunofluorescence staining technology was used to locate the distribution of LSS in cornea,lens and retina tissue.The contents of lanosterol in the cornea,lens and retina tissue were analyzed by liquid chromatograph mass spectrometer (LC-MS).Results No LSS protein and mRNA was expressed in the retinal tissue in normal rats.The mean relative expression of LSS protein in the lens and cornea was 0.43±0.05 and 0.25±0.03,respectively,showing a significant difference between them (t =-5.35,P＜ 0.01).The relative expression of LSS mRNA was 0.51 ±0.04 and 0.29 ±0.02 in the lens and cornea,respectively,with a stronger expression in the lens in comparison with the cornea (t =-8.34,P＜0.01).Immunofluorescence staining showed that LSS primarily located in corneal epithelial layer,stromal layer and endothelial layer as well as lens epithelial cells and shallow cortex layer and hardly expressed in retina,and no co-expression of LSS with the neuron marked by NeuN and the Müller cell marked by glutamine synthetase (GS) in retinal tissue.LC-MS analysis revealed that the contents of lanosterol in lens and cornea was (24.37 ±2.91) ng/mg and (5.31 ±0.58) ng/mg,respectively,with a significant difference between them (t =-11.13,P＜0.01).Conclusions LSS and lanosterol extensively distribute in cornea and lens of normal rats,but not in retina tissue.These results offer new strategies for the target treatment of relevant eye diseases.

Association of the single nucleotide polymorphisms in the calcitonin receptor-like receptor gene with primary angle closure in a Han Chinese population / 国际眼科杂志(Guoji Yanke Zazhi)

?AIM: To study the association of the single nucleotide polymorphism ( SNP) rs1157699 in the calcitonin receptor-like receptor ( CRLR ) gene with primary angle closure ( PAC) in a Han Chinese population.?METHODS: All samples, involved 232 PAC cases and 306 controls, were obtained from an epidemiologic survey conducted in Funing, Jiangsu Province, China. Genotyping were carried out by TaqMan-MGB probe using the real time quantitative polymerase chain reaction system to study the relationship between SNP of rs1157699 in CRLR gene and PAC.?RESULTS: The prevalence of CRLRrs1157699 genotype was 67.4%, 30.0%, 2.6% for CC, CT, TT in cases, and 71.3%, 27.0%, 1.7% in controls respectively.There was no difference between the two groups in the distribution of genotype and allele frequencies of rs1157699 (P>0.05).?CONCLUSION:Our results do not support a significant role for rs1157699 in CRLR with PAC.

Distribution of peroxisome proliferator-activated receptor γ in ocular tissues of rodent animal / 中华实验眼科杂志

Background Peroxisome proliferator-activated receptor gamma (PPARγ) is one of nuclear transcription factors.It plays potential anti-inflammation,anti-fibrogenesis,anti-angiogenesis and neuroprotection roles in human.So the study of its physiological and pathological function in human and animals is still a focus.To understand the distribution of PPARγ in ocular tissues is important for the target treatment of eye diseases.Objective Current study was to investigate the expression of PPARγ in different parts of eye in rodent.Methods Cornea,lens,ciliary,retina and optical nerve were isolated from 6 SPF C57BL/6J mice and 1 SD rat.Western blot assay was used to detect the expressions of PPARγprotein in cornea,lens and retina.Immunohistochemistry was used to locate the distribution of PPARγ protein in cornea,lens,ciliary,retina and optical nerve.Also,the co-expression of PPARγ with glutamine synthetase (GS) (a Müller cell specific marker) and glial fibrillary acidic protein (GFAP)(an astrocyte specific marker) in retina and optic nerve was detected by immunofluorescent double staining.Results Western blot assay showed that PPARγ was expressed in the cornea,lens and retina of the mice.Immunohistochemistry revealed that PPARγ mainly located at corneal epithelium with the strongest staining in the basal cells,but only weak staining was seen in corneal endothelial cells and stroma cells.PPARγ was strongly expressed in epithelial cells and shallow cortex layer of mouse lens.In mouse retina,PPARγ was extensively and richly expressed in retinal ganglion cell layer,inner and outer plexiform layers and inner nuclear layer.In addition,PPARγ was also expressed in the non-pigmented epithelial cells in ciliary body.The co-locations of PPARγexpression with GS in retinal tissue and PPARγ expression with GFAP in optical nerve tissue were found in the mice.Conclusions PPARγis proved to distribute extensively in different ocular tissues.These results offer basis for the target treatment of relevant eye diseases.

Qualitative and quantitative analysis of fluoxetine hydrochloride by 19F NMR / 药学学报

The chemical shift of fluoxetine hydrochloride appears at delta 14.15 in 19F NMR analysis. The delta moved upfield slightly from 14.158 to 14.145 when the concentration of solution became diluted from 2.00 to 0.05 mmol x L(-1). Spiking test was suggested to confirm the existence of the compound for qualitative analysis. 19F NMR detection sensitivity test illustrated that a concentration of 17 mg in 1 L water could be detected while the sample was scanned 500 times with optimum parameters. In quantitative analysis, standard curve of concentration versus fluorine signal intensity was proposed to determine the amount of fluoxetine. Long capillary tube containing trifluoroacetic acid was used as internal standard for the integration measurements and straight line was obtained with good fitting. Direct additions of trifluoroethanol to fluoxetine solutions gave a poorer standard curve.

Determination of leukemia stem cells in childhood acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

The aim of this study was to detect the presence of human AML leukemia stem cells (LSC) in childhood patients with acute leukemia (AL) and analyze the correlation between LSC concentrations and minimal residual disease (MRD) levels in AML cases after remission. The multi-parameter flow cytometry (FCM) and a panel of monoclonal antibody combination were used to detect the AML LSC or AML LSC immunophenotype-identical cell (AML LSC-IPIC) concentrations in childhood AML or ALL leukemia both at new diagnosis and at remission and correlated AML LSC to the MRD levels at different time points after remission. The results indicated that the AML LSC or AML LSC-IPIC concentrations [in average 166 (range 14 - 1459)/100 000 mononuclear cells (MNCs)] in AML at initial diagnosis were significantly higher than those in ALL [7 (range 0 - 560)/100 000 MNCs, p < 0.017] and control [0 (range 0 - 6)/100 000 MNCs, p < 0.017], respectively. The AML LSC concentrations in AML at non-CR were in average 36 (range 5 - 224)/100 000 MNCs. No statistical difference (p > 0.05) was found between the AML LSC or AML LSC-IPIC concentrations in AML (in average 6 (range 0 - 41)/100, 000 MNCs) and ALL [10 (range 0 - 105)/100, 000 MNCs] after CR. The significantly negative correlation was noticed between AML LSC concentrations and MRD levels. It is concluded that the AML LSCs exist in newly diagnosed AML, which are significantly reduced when complete remission has achieved, but the low levels of these populations still remain. The phenotypically similar (CD34(+)CD38⁻CD123(+)) AML LSC populations have also been found in the bone marrow from ALL patients, but their concentrations are not significantly different when CR has achieved. The significantly negative correlation between AML LSC concentrations and MRD levels is observed in AML patients after remission.

Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.</p><p><b>METHODS</b>2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.</p><p><b>RESULTS</b>After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.</p><p><b>CONCLUSION</b>2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.</p>

Establishment of animal model with B lineage acute leukemia in nude mice for evaluation of new therapeutic agents / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish an acute leukemia animal model for testing new therapeutic agents in vivo.</p><p><b>METHODS</b>Nude mice were intraperitoneally injected with 2 mg cyclophosphamide, 24 h later 5 x 10(6) acute B-cell leukemia Nalm-6 cells was inoculated via the tail vein, then monitored daily. When animals were paralyzed or dying, the organs including the liver, spleen, lung, heart, kidney, brain, bone marrow, pancreas, testes were removed and fixed with formalin, examined by routine histopathology.</p><p><b>RESULTS</b>After Nalm-6 cells were inoculated the mean survival of mice were( 19.4+/-0.55)d (n=6). The paralysis of mice was followed by weight loss, bent spines, hogback, cachexia and death. Histopathological examination showed that the tumor cells infiltrated liver, spleen, kidney, lung, meninges, interior cerebrum, the liver and kidney were the most affected organs.</p><p><b>CONCLUSION</b>B lineage acute leukemia animal model has been successfully established in the nude mice, which is suitable for testing new therapeutic agents.</p>

Preparation of the immunotoxin 2E8-norcantharidin and its targeting killing effect in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented immunogenicity to patients capable of inducing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule toxin. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent.</p><p><b>METHODS</b>A monoclonal antibody named 2E8 was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) against CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometry. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made.</p><p><b>RESULTS</b>The purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm-6 cells, while only 0.98% of K562 cells reacted with this antibody. The newly generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the growth of CD19+ Nalm-6 cells (P < 0.001), while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells. Since no significant inhibitory effects were identified among immunotoxin 2E8-NCTD, 2E8 antibody and control groups on CD19(-) K562 cells, a significant targeting effect of the 2E8-NCTD against Nalm-6 cells was confirmed.</p><p><b>CONCLUSIONS</b>The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19+ Nalm-6 leukemia cells in vitro, which provides some experimental data for the further development of this new targeting agent.</p>

Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14 / 中华儿科杂志

<p><b>OBJECTIVE</b>Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.</p><p><b>METHODS</b>Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).</p><p><b>RESULTS</b>The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.</p><p><b>CONCLUSIONS</b>The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.</p>

Construction and expression of prokaryotic vector of single chain antibody derived from a new clone of anti-CD14 antibody ZCH-7-2F9 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.</p><p><b>METHODS</b>Primers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.</p><p><b>RESULT</b>The cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.</p><p><b>CONCLUSION</b>The ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.</p>

Diagnosis and surgical treatment of giant intrathoracic solid tumors / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To summarize the experience in diagnosis and surgical treatment of giant intrathoracic solid tumors.</p><p><b>METHODS</b>The data of surgically treated 36 patients with giant intrathoracic solid tumors were analyzed, including 19 males and 17 females. Complete resection was achieved in 34 cases with superior vena cava angioplasty in 3 cases and ligation of the left anonymous vein in 2 cases. Six patients received postoperative radiotherapy.</p><p><b>RESULTS</b>The symptoms in 32 cases were significantly improved. Two patients (5.6%) died of postoperative respiratory infection and failure. The mean postoperative hospital stay was 14.2 days. Pulmonary edema occurred in 6 cases due to rapid decompression of the lung. Pathological results showed that 25 cases had benign tumors and 11 had malignancy. During the follow-up of 1 to 22 years, all patients with benign tumors were still alive, but the patients with malignant tumors had a mean survival time of only 2.1 years.</p><p><b>CONCLUSION</b>Surgical treatment for giant intrathoracic solid tumors is suggested whenever technically possible. Even though a tumor can not be completely resected, satisfied results could still be achieved if combined with postoperative radiotherapy. Proper anesthesia, satisfied exposure with a suitable incision, appropriate resection pattern and hemostatic method are the keys for successful surgical treatment.</p>

Long-term follow-up of treatment outcome and prognosis on 46 children with acute promyelocytic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Acute promyelocytic leukemia (APL) is a specific type of hematopoietic malignancy, accounting for 10% of the de novo acute myeloid leukemia (AML). The data on long-term outcome of APL in children are limited. The aim of this study was to investigate the clinical biological features, diagnosis, prognosis and long-term survival of childhood APL.</p><p><b>METHODS</b>A total of 46 children with newly diagnosed APL from April 1998 to October 2005 were enrolled into this study. Induction treatment containing all-trans retinoic acid (ATRA) plus daunorubicin (DNR) or pirarubicin (THP) was performed on these patients, followed by 6 courses of chemotherapy consolidation: DNR, homoharringtonine or etoposide plus Ara-C. A maintenance therapy was then administered once 3-6 months. The total period of treatment was 2.5 years.</p><p><b>RESULTS</b>Of the 39 patients who had completed the regular treatment, 36 (92.3%) achieved a complete remission. The 5-year cumulative incidence of relapse (CIR) was 28.6%. The estimated overall survival (OS) rates at 1, 3 and 5 years were (86.1 +/- 5.8)%, (76.1 +/- 7.5)% and (70.2 +/- 8.9)% respectively, while the event free survival (EFS) rates were (78.4 +/- 6.8)%, (63.6 +/- 8.7)% and (53.1 +/- 10.0)% respectively. The 5-year OS rate of patients with WBC less than or equal to 10.0 X 10(9)/L was (81.4 +/- 10.3)%, which was significantly higher than that with WBC greater than 10.0 X 10(9)/L[(51.6 +/- 14.7)%, P < 0.05]. Five patients with RT-PCR positive for PML/RARalpha S (short) subtype died eventually although all of them achieved CR, but none of the 13 patients with PML/RARalpha L (long) subtype died.</p><p><b>CONCLUSIONS</b>Remission induction therapy with ATRA + DNR or THP is effective and safe for newly diagnosed childhood APL. The remission induction therapy combined with chemotherapy containing high/intermediate dose Ara-C can improve the long-term survival rates of APL patients. High WBC count and S subtype of PML-RARa are two poor prognostic factors for children with APL.</p>

Evaluation of the P-gp pump function on leukemic cell membrane and proper application of its reversal agents with Calcein-AM and flow cytometry / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common malignancy in children. Combined chemotherapy is currently the primary treatment modality. During the past decade, very high cure rates of childhood acute lymphoblastic leukemia (ALL) have been reported both at home and abroad. However, the cure rates of children with acute myeloid leukemia (AML) remain low due to the multiple-drug resistance (MDR). P-glycoprotein (P-gp) is one of the most important mechanisms of MDR for leukemia cells. However, the function of the protein, the clinical application of its reversal agents and the efficacy of the combination of the reversal agents remain to be elucidated. The present study aimed to evaluate the P-gp pump function on leukemia cell membrane and the effects of the combined administration of the reversal agents cyclosporin A (CSA) and verapamil (VER) through the observation of Calcein-AM (C-AM) metabolism in the cell line K562 and its multi-drug resistant subline K562/VCR.</p><p><b>METHODS</b>The mean fluorescence intensity (MFI) of C-AM inside the cytoplasm was analyzed with flow cytometry (FCM). The events of K562 and K562/VCR cells treated and untreated with CSA, VER and CSA + VER were acquired at time points 0, 30, 60, 90 and 120 minutes, respectively, and the data obtained were analyzed with CellQuest software.</p><p><b>RESULTS</b>The C-AM in the K562 and K562/VCR varied more apparently in the fist 24 hours. In addition, the MFI of the C-AM in K562 was significantly higher than that in K562/VCR cells indicating that the P-gp pump molecules were functioning. The MFIs of the CSA, VER and CSA + VER groups co-cultured with K562/VCR cells were 4014 +/- 219, 3879 +/- 116 and 4158 +/- 302, respectively after 120 min of incubation, significantly higher as compared to that of control group (3251 +/- 107, P < 0.05). On the other hand, significant inhibition of the efflux from the K562/VCR cell line was also noticed after the same time period of incubation with the MFIs of 2237 +/- 155, 1932 +/- 233 and 2231 +/- 147, respectively in the three groups, which was significantly higher than that of control group (1622 +/- 191, P < 0.05). CSA, VER and CSA + VER could increase the uptake and inhibit the efflux of C-AM by K562/VCR cells, while no evident influence on those functions inside the parental cell line K562 cells was noticed.</p><p><b>CONCLUSIONS</b>CSA, VER and CSA + VER could increase the uptake and reduce the efflux of C-AM by K562/VCR cells while no significant difference between the CSA + VER and CSA or VER was noticed. P-gp pump function and the effects of its reversal agents on leukemic cells can be rapidly and easily evaluated by using the C-AM and FCM.</p>